Field of the invention
[0001] The present invention relates to cationic lipid/protein/nucleic acid complexes comprising
viral packaging proteins and their use in the efficient delivery of nucleic acids
to cells, such as neuronal cells.
Background to the invention
[0002] Promising advances in non-viral gene transfer have been made as a result of the production
of synthetic liposomes formulated with cationic lipids that are able to transfect
cells. However few of these complexes have been examined for their ability to efficiently
transfer DNA into CNS cells and to obtain expression of a transgene. The ability to
transfect neuronal cells efficiently and safely could provide a powerful tool for
the elucidation of neuronal function and may lead to novel treatments for neurological
disorders.
[0003] Unfortunately, gene therapy for the CNS has been hampered by the lack of efficient
means for transducing postmitotic neurons. Most studies have utilized viral vectors
for gene delivery. However, many viral vectors are plagued by problems of immunity
and cytotoxicity and are not easily manipulated by non-virologists
1-3. Non-viral vectors are now emerging as an alternative method of cellular transduction.
The most promising advances in non-viral gene transfer have been in the production
of synthetic liposomes formulated with cationic lipids (cytofectins) able to transfect
cells. Such cationic liposomes are relatively easy to use, have a broad applicability
and lack cytotoxicity
4.
[0004] Novel cationic liposome formulations are constantly being developed
5. However, few of these complexes have been examined for their ability to efficiently
transduce cells within the CNS
6-9. Cationic liposomes act via electrostatic interactions with negatively charged DNA
and subsequently with cellular membranes where they are taken across the cell membrane
by a process of slow endocytosis 6, 10, 11. They are frequently formulated using the
neutral lipid dioleoyl-L-α-phosphatidylethanolamine (DOPE), which is extremely efficient
at endosomal buffering and disruption
8,
12. From the perinuclear space transfected genetic material is released from the liposome
complex, transported to the nucleus and expressed. To date only liposomes formulated
from N- [1-(2,3-dioleyloxy)propyl]-N,N,N trimethyl ammonium chloride (DOTMA) and DOPE,
have been shown to mediate successful transfection in the CNS
13-16. To be useful for gene therapy liposome complexes capable of transfecting CNS cells
with high efficiency are needed.
[0005] A major limitation in non-viral mediated gene transfer is the formation of large
aggregated molecules during the generation of liposome:DNA complexes
5. These large aggregates may reduce the efficiency of transfection possibly by limiting
endocytosis of the complexes. One approach to circumvent this is to reduce the size
of DNA molecules via DNA condensation prior to complex formation. Pre-condensation
of DNA produces smaller complexes and improved transfection efficiencies
17-23. Various polycations have been identified which are efficient at improving liposome-mediated
transfections. Of these, poly-L-lysine and protamine have produced the most dramatic
results enabling increases of over 30 fold compared to complexes without pre-condensation
in a variety of non-neuronal cell lines 17, 21.
[0006] Protamine sulphate is particularly good at enhancing liposomal transfection. Protamine
is a naturally occurring polycation found in the head of spermatozoa. The role of
protamine is to condense DNA in sperm and aid in its transfer to the egg nucleus.
The nuclear targeting property of protamine makes it particularly attractive for gene
transfer. Also, unlike the synthetic poly-L-lysine, which has a range of large molecular
weights (18000-19200 Da), protamine is naturally occurring, smaller and more uniform
in size (4000-4250 Da). These qualities mean there is less chance for immunogenic
responses in the target tissue and the condensation is easier to control. Other naturally
occurring DNA condensing proteins have also been used to enhance cationic liposome
mediated DNA transfer. Fritz et al,
22 achieved approximately 30 fold increases in lipofection using a recombinant human
H1 histone protein incorporating a nuclear localization signal (nls-Hl). Also, the
non-histone chromosomal high mobility group 1,2 protein has been shown to improve
lipofection and is used routinely in the HVJ-liposome method
20,24.
Summary of the invention
[0007] We have examined viral-DNA associated proteins for their ability to improve liposome
based gene transfer. In particular we have compared the viral-coded synthetic peptide
Mul and recombinant Vp1 protein of adenovirus and polyomavirus respectively. Mul may
play a role in adenoviral chromosome condensation while VP1 is the only structural
protein of polyomavirus to exhibit DNA binding activity
25-27. Vp1, but not Mu1 contains an embedded classical nuclear localization signal (NLS)
similar to that found in HMG-1,2 and nls-Hl
26. We found that Mu1, but not Vp1, significantly improved cationic liposome mediated
gene transfer in cells derived from the nervous system and kidney. We also found that
Mu1 enhancement was greater in differentiated cells indicating the possible usefulness
of this approach for neuronal cells
in vivo.
[0008] These findings have implications for experimental and therapeutic uses of liposome-mediated
delivery of DNA to CNS cells.
[0009] Accordingly, the present invention provides a non-viral nucleic acid delivery vector
comprising a condensed polypeptide/ nucleic acid complex and a cationic lipid, wherein
the complex comprises
(a) a nucleic acid sequence of interest (NOI); and
(b) one or more viral nucleic acid packaging polypeptides, or derivatives thereof,
said polypeptides or derivatives thereof being (i) capable of binding to the NOI;
and (ii) capable of condensing the NOI; and wherein the NOI is heterologous to the
polypeptide.
[0010] Preferably, at least one polypeptide is an adenoviral nucleic acid packaging polypeptide,
or derivative thereof. More preferably, the adenoviral polypeptide is Mu1, pV or pVII
or a derivative thereof.
[0011] The term "heterologous to the polypeptide" means that viral NOIs that naturally occur
in combination with the viral packaging polypeptide are excluded.
[0012] In a preferred embodiment, the vector further comprises a polypeptide comprising
a nuclear localisation sequence (NLS). More preferably, the polypeptide comprising
a nuclear localisation sequence (NLS) is adenoviral pV or a derivative thereof.
[0013] The present invention also provides a condensed polypeptide/nucleic acid complex
comprising a cationic lipid, a polypeptide component and a nucleic acid component,
for use in delivering the nucleic acid component to a nucleus of a eukaryotic cell,
wherein
(i) the polypeptide component is a viral nucleic acid packaging polypeptide, or derivative
thereof;
(ii) the polypeptide component or derivative thereof is capable of binding to the
NOI; and
(iii) the polypeptide component or derivative thereof is capable of condensing the
NOI; and wherein the nucleic acid is heterologous to the polypeptide.
[0014] Preferably, at least one polypeptide is an adenoviral nucleic acid packaging polypeptide,
or derivative thereof. More preferably, the adenoviral polypeptide is Mu1, pV or pVII
or a derivative thereof.
[0015] In a preferred embodiment, the complex further comprises a polypeptide comprising
a nuclear localisation sequence (NLS). More preferably, the polypeptide comprising
a nuclear localisation sequence (NLS) is adenoviral pV or a derivative thereof.
[0016] The present invention also provides a method of producing a non-viral nucleic acid
delivery vector comprising a condensed polypeptide/ nucleic acid complex and a cationic
lipid, which method comprises
(a) contacting an nucleic acid sequence of interest (NOI) with a viral nucleic acid
packaging polypeptide or derivative thereof, said polypeptide component or derivative
thereof being (i) capable of binding to the NOI; and (ii) capable of condensing the
NOI; and wherein the NOI is heterologous to the polypeptide; and
(b) contacting the nucleic acid/polypeptide complex thus formed with a cationic lipid.
[0017] The present invention further provides a method of introducing a nucleic acid sequence
of interest (NOI) into a eukaryotic cell which method comprises contacting the cell
with a complex of the invention wherein the complex comprises the NOI. Preferably
the cell is a neuronal, cancer or epithelial cell.
[0018] In an alternative embodiment, a viral nucleic acid nuclear localisation/delivery
polypeptide may be used instead of, or in addition to a viral nucleic acid packaging
polypeptide. Indeed, some viral polypeptides combine both functions.
Detailed description of the invention
[0019] Although in general the techniques mentioned herein are well known in the art, reference
may be made in particular to Sambrook
et al., Molecular Cloning, A Laboratory Manual (1989) and Ausubel
et al., Short Protocols in Molecular Biology (1999) 4
th Ed, John Wiley & Sons, Inc.
A. Polypeptide components
1. Viral nucleic acid packaging polypeptides
[0020] The term "viral nucleic acid packaging polypeptides" typically includes polypeptides
encoded by viral genomes that occur naturally in viral particles where their function
is to package, in particular condense, and deliver into the nucleus the nucleic acids
constituting the viral genome into the virion. Also included are homologues and derivatives
thereof, such as fragments, as discussed below.
[0021] Examples of viral nucleic acid packaging polypeptides include viral core proteins
such as hepatitis B core antigen and adenoviral core proteins, Mu1, pV and pVII and
their equivalents proteins in other adenoviruses, such as Mastadenoviruses (mammalian
adenoviruses) and Aviadenoviruses (bird adenoviruses). A particularly preferred viral
nucleic acid packaging polypeptide for use in the present invention is the Mu1 polypeptide
shown immediately below as SEQ I.D. No. 1.
[0022] A viral nucleic acid packaging polypeptide for use in the present invention is capable
of binding to nucleic acids, typically in a non-specific manner, preferably causing
condensation of the nucleic acid. It is generally preferred that the condensed NOl
has a size of equal to or less than 200 nm, such as from 50 to 200 nm, for optimal
efficiency of delivery to a target cell.
[0023] The ability of viral polypeptides to bind to nucleic acids may be determined
in vitro using techniques such as gel electrophoresis including gel retardation assays (see
materials and methods section and results section) and electrophoretic band shift
mobility assays, ethidium bromide exclusion assays and affinity chromatography (for
example using single-or double-stranded DNA cellulose).
[0024] The ability of viral polypeptides to condense nucleic acids may be determined by,
for example, circular dichroism (CD) spectroscopy (see, for example, Sato and Hosokawa,
1984, J. Biol. Chem. 95: 1031-1039).
[0025] Generally the viral polypeptides, or homologues or derivatives thereof, will comprise
a number of positively charged amino acid residues at physiological pH (such as pH
7.4). Preferably the overall net charge on the viral polypeptide is positive at physiological
pH. In particular, it is preferred that the charge : amino acid ratio is at least
+0.3, preferably at least +0.4, +0.5 or +0.6.
[0026] It is preferred that the viral polypeptides, or homologues or derivatives thereof
comprise arginine residues rather than lysine residues or a mixture of both. It is
also particularly preferred that the viral polypeptides, or homologues or derivatives
thereof comprise one or more histidine residues, preferably two or more histidine
residues. In addition, the viral polypeptides, or homologues or derivatives thereof
will typically comprise a number of highly hydrophobic residues, such as alanine,
for example two or more hydrophobic residues.
[0027] It will be understood that amino acid sequences for use in the invention are not
limited to naturally occurring viral nucleic acid packaging polypeptides but also
include homologous sequences obtained from any source, for example related viral/bacterial
proteins, cellular homologues and synthetic peptides, as well as variants or derivatives,
such as fragments, thereof.
[0028] In the context of the present invention, a homologous sequence is taken to include
an amino acid sequence which is at least 60, 70, 80 or 90% identical, preferably at
least 95 or 98% identical at the amino acid level over at least 10 preferably at least
20, 30, 40 or 50 amino acids with a viral core polypeptide, for example the Mu1 sequence
shown as SEQ I.D. No. 1. In particular, homology should typically be considered with
respect to those regions of the sequence known to be essential for nucleic acid binding
rather than non-essential neighbouring sequences. Although homology can also be considered
in terms of similarity (i.e. amino acid residues having similar chemical properties/functions),
in the context of the present invention it is preferred to express homology in terms
of sequence identity.
[0029] Homology comparisons can be conducted by eye, or more usually, with the aid of readily
available sequence comparison programs. These commercially available computer programs
can calculate % homology between two or more sequences.
[0030] % homology may be calculated over contiguous sequences, i.e. one sequence is aligned
with the other sequence and each amino acid in one sequence directly compared with
the corresponding amino acid in the other sequence, one residue at a time. This is
called an ''ungapped'' alignment Typically, such ungapped alignments are performed
only over a relatively short number of residues (for example less than 50 contiguous
amino acids).
[0031] Although this is a very simple and consistent method, it fails to take into consideration
that, for example, in an otherwise identical pair of sequences, one insertion or deletion
will cause the following amino acid residues to be put out of alignment, thus potentially
resulting in a large reduction in % homology when a global alignment is performed.
Consequently, most sequence comparison methods are designed to produce optimal alignments
that take into consideration possible insertions and deletions without penalising
unduly the overall homology score. This is achieved by inserting "gaps" in the sequence
alignment to try to maximise local homology.
[0032] However, these more complex methods assign "gap penalties" to each gap that occurs
in the alignment so that, for the same number of identical amino acids, a sequence
alignment with as few gaps as possible - reflecting higher relatedness between the
two compared sequences - will achieve a higher score than one with many gaps. "Affine
gap costs" are typically used that charge a relatively high cost for the existence
of a gap and a smaller penalty for each subsequent residue in the gap. This is the
most commonly used gap scoring system. High gap penalties will of course produce optimised
alignments with fewer gaps. Most alignment programs allow the gap penalties to be
modified. However, it is preferred to use the default values when using such software
for sequence comparisons. For example when using the GCG Wisconsin Bestfit package
(see below) the default gap penalty for amino acid sequences is -12 for a gap and
-4 for each extension.
[0033] Calculation of maximum % homology therefore firstly requires the production of an
optimal alignment, taking into consideration gap penalties. A suitable computer program
for carrying out such an alignment is the GCG Wisconsin Bestfit package (University
of Wisconsin, U.S.A.; Devereux
et al., 1984, Nucleic Acids Research 12:387). Examples of other software than can perform
sequence comparisons include, but are not limited to, the BLAST package (see
Ausubel et al., 1999
ibid - Chapter 18), FASTA (Atschul
et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST
and FASTA are available for offline and online searching (see Ausubel
et al., 1999
ibid, pages 7-58 to 7-60). However it is preferred to use the GCG Bestfit program.
[0034] Although the final % homology can be measured in terms of identity, the alignment
process itself is typically not based on an all-or-nothing pair comparison. Instead,
a scaled similarity score matrix is generally used that assigns scores to each pairwise
comparison based on chemical similarity or evolutionary distance. An example of such
a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite
of programs. GCG Wisconsin programs generally use either the public default values
or a custom symbol comparison table if supplied (see user manual for further details).
It is preferred to use the public default values for the GCG package, or in the case
of other software, the default matrix, such as BLOSUM62.
[0035] Once the software has produced an optimal alignment, it is possible to calculate
% homology, preferably % sequence identity. The software typically does this as part
of the sequence comparison and generates a numerical result.
[0036] The terms "derivative" in relation to the amino acid sequences used in the present
invention includes any substitution of, variation of, modification of, replacement
of, deletion of or addition of one (or more) amino acids from or to the sequence providing
the resultant amino acid sequence has nucleic acid binding and condensation activity,
preferably having at least the same activity as the unmodified polypeptides.
[0037] Viral polypeptides may be modified for use in the present invention. Typically, modifications
are made that maintain the nucleic acid binding and condensation properties of the
sequence. Amino acid substitutions may be made, for example from 1, 2 or 3 to 10,
20 or 30 substitutions provided that the modified sequence retains nucleic acid binding
and condensation properties. Amino acid substitutions may include the use of non-naturally
occurring analogues, for example to increase blood plasma half-life of a therapeutically
administered polypeptide.
[0038] In particular, it may be desirable to make amino acid substitutions to increase the
net positive charge, at physiological pH, of a naturally occurring viral packaging
polypeptide. Positively charged amino acids include arginine, lysine and histidine.
Arginine is the most highly charged of the naturally occurring amino acids and is
particularly preferred.
ALIPHATIC |
Non-polar |
G A P |
IL V |
Polar - uncharged |
C S T M |
N Q |
Polar - charged |
D E |
K R |
AROMATIC |
|
H F W Y |
[0039] Conservative substitutions may be made, for example according to the Table above.
Amino acids in the same block in the second column and preferably in the same line
in the third column may be substituted for each other:
[0040] Polypeptides for use in the invention may be made by recombinant means, for example
as described below. However they may also be made by synthetic means using techniques
well known to skilled persons such as solid phase synthesis. Polypeptides for use
in the invention may also be produced as fusion proteins, for example to aid in extraction
and purification. Examples of fusion protein partners include glutathione-S-transferase
(GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and β-galactosidase.
It may also be convenient to include a proteolytic cleavage site between the fusion
protein partner and the protein sequence of interest to allow removal of fusion protein
sequences. Preferably the fusion protein partner will not hinder the biological activity
of the protein of interest sequence.
[0041] Polypeptides for use in the invention may be in a substantially isolated form. It
will be understood that the polypeptides may be mixed with carriers or diluents which
will not interfere with the intended purpose of the polypeptides and still be regarded
as substantially isolated. The polypeptides may also be in a substantially purified
form, in which case generally more than 90%, e.g. 95%, 98% or 99% of the protein in
the preparation comprises polypeptides for use in the invention.
2. Polypeptides comprising nuclear localisation sequences
[0042] In a preferred embodiment, the delivery vector/complex of the invention further comprises
a polypeptide comprising a nuclear localisation sequence (NLS). In general, NLSs are
well known in the art (see, for example, Dingwall and Laskey, 1991, Trends. Biochem.
Sci. 16: 478-481). However, it is particularly preferred to use the NLS of adenovirus
core , protein pV. The NLS of pV has the sequence RPRRRATTRRRTTTGTRRRRRRR (SEQ I.D.
No.2) corresponding to amino acids 315-337 (D. Matthews, submitted.) A further NLS
is present in the N-terminus (KPRKLKRVKKKKK - SEQ I.D. No. 3), although the C-terminal
NLS is preferred.
[0043] The NLS may be present on a separate polypeptide molecule to the packaging polypeptide
or as part of the same polypeptide chain, for example in a fusion protein.
B. Nucleic acid sequences of interest
[0044] Nucleic acid sequences of interest (NOIs) intended to be delivered to cells using
the delivery vector or complex of the invention may comprise DNA or RNA. They may
be single-stranded or double-stranded. They may also be polynucleotides which include
within them synthetic or modified nucleotides. A number of different types of modification
to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate
backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the
molecule. For the purposes of the present invention, it is to be understood that the
polynucleotides described herein may be modified by any method available in the art.
Such modifications may be carried out in order to enhance the
in vivo activity or life span of the NOIs.
[0045] The NOI typically comprises a heterologous gene. The term ''heterologous gene" encompasses
any gene, The heterologous gene may be any allelic variant of a wild-type gene, or
it may be a mutant gene. The term "gene" is intended to cover nucleic acid sequences
which are capable of being at least transcribed. Thus, sequences encoding mRNA, tRNA
and rRNA, as well as antisense constructs, are included within this definition. Nucleic
acids may be, for example, ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or
analogues thereof. Sequences encoding mRNA will optionally include some or all of
5' and/or 3' transcribed but untranslated flanking sequences naturally, or otherwise,
associated with the translated coding sequence. It may optionally further include
the associated transcriptional control sequences normally associated with the transcribed
sequences, for example transcriptional stop signals, polyadenylation sites and downstream
enhancer elements.
[0046] The transcribed sequence of the heterologous gene is preferably operably linked to
a control sequence permitting expression of the heterologous gene in mammalian cells,
preferably neuronal cells, such as cells of the central and peripheral nervous system,
cancer or epithelial cells. The term "operably linked" refers to a juxtaposition wherein
the components described are in a relationship permitting them to function in their
intended manner. A control sequence "operably linked" to a coding sequence is ligated
in such a way that expression of the coding sequence is achieved under conditions
compatible with the control sequence.
[0047] The control sequence comprises a promoter allowing expression of the heterologous
gene and a signal for termination of transcription. The promoter is selected from
promoters which are functional in mammalian, preferably human cells. The promoter
may be derived from promoter sequences of eukaryotic genes. For example, it may be
a promoter derived from the genome of a cell in which expression of the heterologous
gene is to occur, preferably a cell of the mammalian central or peripheral nervous
system. With respect to eukaryotic promoters, they may be promoters that function
in a ubiquitous manner (such as promoters of β-actin, tubulin) or, alternatively,
a tissue-specific manner (such as promoters of the genes for pyruvate kinase). They
may also be promoters that respond to specific stimuli, for example promoters that
bind steroid hormone receptors. Viral promoters may also be used, for example the
Moloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter or promoters
of herpes virus genes.
[0048] It may also be advantageous for the promoters to be inducible so that the levels
of expression of the heterologous gene can be regulated during the life-time of the
cell. Inducible means that the levels of expression obtained using the promoter can
be regulated.
[0049] In addition, any of these promoters may be modified by the addition of further regulatory
sequences, for example enhancer sequences. Chimeric promoters may also be used comprising
sequence elements from two or more different promoters described above. Furthermore,
the use of locus control regions (LCRs) may be desirable.
[0050] The heterologous gene will typically encode a polypeptide of therapeutic use. In
accordance with the present invention, suitable NOI sequences include those that are
of therapeutic and/or diagnostic application such as, but are not limited to: sequences
encoding cytokines, chemokines, hormones, antibodies, engineered immunoglobulin-like
molecules, a single chain antibody, fusion proteins, enzymes, immune co-stimulatory
molecules, immunomodulatory molecules, anti-sense RNA, a transdominant negative mutant
of a target protein, a toxin, a conditional toxin, an antigen, a tumour suppressor
protein and growth factors, membrane proteins, vasoactive proteins and peptides, anti-viral
proteins and ribozymes, and derivatives therof (such as with an associated reporter
group).
[0051] Examples of polypeptides of therapeutic use include neurotrophic factors such as
nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), brain-derived neurotrophic
factor (BNTF) and neurotrophins (such as NT-3, NT-4/5) which have potential as therapeutic
agents for the treatment of neurological disorders such as Parkinson's disease.
[0052] Suitable NOIs for use in the present invention in the treatment or prophylaxis of
cancer include NOIs encoding proteins which: destroy the target cell (for example
a ribosomal toxin), act as: tumour suppressors (such as wild-type p53); activators
of anti-tumour immune mechanisms (such as cytokines, co-stimulatory molecules and
immunoglobulins); inhibitors of angiogenesis; or which provide enhanced drug sensitivity
(such as pro-drug activation enzymes); indirectly stimulate destruction of target
cell by natural effector cells (for example, strong antigen to stimulate the immune
system or convert a precursor substance to a toxic substance which destroys the target
cell (for example a prodrug activating enzyme). Encoded proteins could also destroy
bystander tumour cells (for example with secreted antitumour antibody-ribosomal toxin
fusion protein), indirectly stimulated destruction of bystander tumour cells (for
example cytokines to stimulate the immune system or procoagulant proteins causing
local vascular occlusion) or convert a precursor substance to a toxic substance which
destroys bystander tumour cells (eg an enzyme which activates a prodrug to a diffusible
drug).
[0053] NOI(s) may be used which encode antisense transcripts or ribozymes which interfere
with the expression of cellular or pathogen genes, for example, with expression of
cellular genes for tumour persistence (for example against aberrant
myc transcripts in Burkitts lymphoma or against
bcr-abl transcripts in chronic myeloid leukemia. The use of combinations of such NOIs is
also envisaged.
[0054] Instead of, or as well as, being selectively expressed in target tissues, the NOI
or NOIs may encode a pro-drug activation enzyme or enzymes which have no significant
effect or no deleterious effect until the individual is treated with one or more pro-drugs
upon which the enzyme or enzymes act In the presence of the active NOI, treatment
of an individual with the appropriate pro-drug leads to enhanced reduction in tumour
growth or survival.
[0055] A pro-drug activating enzyme may be delivered to a tumour site for the treatment
of a cancer. In each case, a suitable pro-drug is used in the treatment of the patient
in combination with the appropriate pro-drug activating enzyme. An appropriate pro-drug
is administered in conjunction with the vector. Examples of pro-drugs include: etoposide
phosphate (with alkaline phosphatase); 5-fluorocytosine (with cytosine deaminase);
doxorubicin-N-p-hydroxyphenoxyacetamide (with penicillin-V-amidase); para-N-bis(2-chloroethyl)
aminobenzoyl glutamate (with carboxypeptidase G2); cephalosporin nitrogen mustard
carbamates (with β-lactamase); SR4233 (with P450 Reducase); ganciclovir (with HSV
thymidine kinase); mustard pro-drugs with nitroreductase and cyclophosphamide (with
P450).
[0056] Examples of suitable pro-drug activation enzymes for use in the invention include
a thymidine phosphorylase which activates the 5-fluoro-uracil pro-drugs capcetabine
and furtulon; thymidine kinase from herpes simplex virus which activates ganciclovir;
a cytochrome P450 which activates a pro-drug such as cyclophosphamide to a DNA damaging
agent; and cytosine deaminase which activates 5-fluorocytosine. Preferably, an enzyme
of human origin is used
[0057] NOIs may also encode antigenic polypeptides for use as vaccines. Preferably such
antigenic polypeptides are derived from pathogenic organisms, for example bacteria
or viruses. Examples of such antigenic polypeptides include hepatitis C virus antigens,
hepatitis B surface or core antigens, HIV antigens, pertussis toxin, cholera toxin
or diphtheria toxin.
[0058] NOIs may also include marker genes (for example encoding β-galactosidase or green
fluorescent protein) or genes whose products regulate the expression of other genes
(for example, transcriptional regulatory factors).
[0059] Where a disease is caused by a defective gene, NOIs may be admistered that encode
a fully functional allele of the gene, such as in the case of cystic fibrosis. The
molecular basis for a variety of genetic disorders has been identified and wild type
functional sequences cloned. It may be desirable to include in the NOI flanking sequences
to the therapeutic gene that are homologous to the corresponding flanking sequences
in the genome to allow for replacement of the defective gene by homologous recombination.
[0060] Gene therapy and other therapeutic applications may well require the administration
of multiple genes. The expression of multiple genes may be advantageous for the treatment
of a variety of conditions. Since there is no limitation in the size of NOI that may
be incorporated into a delivery vector or complex of the invention, it should be possible
to target cells with multiple genes simultaneously.
C. Cationic lipids
[0061] A variety of cationic lipids is known in the art - see for example WO95/02698, the
disclosure of which is herein incorporated by reference, some of which is reproduced
below. Example structures of cationic lipids useful in this invention are provided
in Table 1 of WO95/02698. Generally, any cationic lipid, either monovalent or polyvalent,
can be used in the compositions and methods of this invention. Polyvalent cationic
lipids are generally preferred. Cationic lipids include saturated and unsaturated
alkyl and alicyclic ethers and esters of amines, amides or derivatives thereof. Straight-chain
and branched alkyl and alkene groups of cationic lipids can contain from 1 to about
25 carbon atoms. Preferred straight-chain or branched alkyl or alkene groups have
six or more carbon atoms. Alicyclic groups can contain from about 6 to 30 carbon atoms.
Preferred alicyclic groups include cholesterol and other steroid groups. Cationic
lipids can be prepared with a variety of counterions (anions) including among others:
chloride, bromide, iodide, fluoride, acetate, trifluoroacetate, sulfate, nitrite,
and nitrate.
[0062] A well-known cationic lipid is N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium
chloride (DOTMA).
[0063] DOTMA and the analogous diester DOTAP (1,2-bis(oleoyloxy)-3 (trimethylammonium) propane),
are commercially available. Additional cationic lipids structurally related to DOTMA
are described in U.S. Patent 4,897,355, which is herein incorporated by reference.
[0064] Another useful group of cationic lipids related to DOTMA and DOTAP are commonly called
DORI-ethers or DORI-esters. DORI lipids differ from DOTMA and DOTAP in that one of
the methyl groups of the trimethylammonium group is replaced with a hydroxyethyl group.
The oleoyl groups of DORI lipids can be replaced with other alkyl or alkene groups,
such as palmitoyl or stearoyl groups. The hydroxyl group of the DORI-type lipids can
be used as a site for further functionalization, for example for esterification to
amines, like carboxyspermine.
[0065] Additional cationic lipids which can be employed in the delivery vectors or complexes
of this invention include those described in WO91/15501 as useful for the transfection
of cells.
[0066] Cationic sterol derivatives, like 3β[N-(N',N'- dimethylaminoethane)carbamoyl] cholesterol
(DC-Chol) in which cholesterol is linked to a trialkyammonium group, can also be employed
in the present invention. DC-Chol is reported to provide more efficient transfection
and lower toxicity than DOTMA-containing liposomes for some cell lines. DC-Chol polyamine
variants such as those described in WO97/45442 may also be used.
[0067] Polycationic lipids containing carboxyspermine are also useful in the delivery vectors
or complexes of this invention. EP-A-304111 describes carboxyspermine containing cationic
lipids including 5-carboxyspermylglycine dioctadecyl-amide (DOGS) and dipalmitoylphosphatidylethanolamine
5-carboxyspermylamide (DPPES). Additional cationic lipids can be obtained by replacing
the octadecyl and palmitoyl groups of DOGS and DPPES, respectively, with other alkyl
or alkene groups.
[0068] In the delivery vectors or complexes of the invention cationic lipids can optionally
be combined with non-cationic co-lipids, preferably neutral lipids, to form liposomes
or lipid aggregates. Neutral lipids useful in this invention include, among many others:
lecithins; phosphatidylethanolamines, such as DOPE (dioleoyl phosphatidylethanolamine),
POPE (palinitoyloleoylphosphatidylethanolamine) and DSPE (distearoylphosphatidylethanol
amine); phosphatidylcholine; phosphatidylcholines, such as DOPC (dioleoyl phosphatidylcholine),
DPPC (dipalmitoylphosphatidylcholine) POPC (palmitoyloleoyl phosphatidylcholine) and
DSPC (distearoylphosphatidylcholine); phosphatidylglycerol; phospha- tidylglycerols,
such as DOPG (dioleoylphosphatidylglycerol), DPPG (dipalmitoylphosphatidylglycerol),
and DSPG (distearoylphosphatidylglycerol); phosphatidylserines, such as dioleoyl-
or dipalmitoylphospatidylserine; diphospha tidylglycerols; fatty acid esters; glycerol
esters; sphingolipids; cardolipin; cerebrosides; and ceramides; and mixtures thereof.
Neutral lipids also include cholesterol and other 3DOH-sterols.
[0069] Moreover in the delivery vector or complexes of the invention one or more amphiphilic
compounds can optionally be incorporated in order to modify its surface property.
Amphiphilic compounds useful in this invention include, among many others; neoglycolipids
such as GLU4 and GLU7 shown in Figure 22, polyethyleneglycol lipids such as N-(ω-methoxy(polyoxyethylene)oxycarbonyl)-phosphatidylethanolamine,
N-monomethoxy(polyoxyethylene)succinylphosphatidylethanol-amine and polyoxyethylene
cholesteryl ether; nonionic detergents such as alkyl glycosides, alkyl methyl glucamides,
sucrose esters, alkyl polyglycerol ethers, alkyl polyoxyethylene ethers and alkyl
sorbitan oxyethylene ethers and steroidal oxyethylene ethers; block copolymers such
as polyoxyethylene polyoxypropylene block copolymers.
[0070] In one aspect the cationic lipid of the present invention is modified with a sugar
moiety or a polyethylene glycol (PEG) moiety. In a further aspect the complex of the
invention further comprises a compound capable of acting as a cationic lipid, the
compound comprising a cholesterol group having linked thereto via an amine group,
a sugar moiety or a polyethylene glycol moiety. As demonstrated in the Examples we
have found such sugar/PEG modified cationic lipids to be particularly advantageous.
Thus in a further aspect the present invention provides a compound capable of acting
as a cationic lipid, the compound comprising a cholesterol group having linked thereto
via an amine group, a sugar moiety or a polyethylene glycol moiety. Preferably the
compound comprises from 1 to 7 sugar moieties or a polyethylene glycol moieties. The
compound may comprise a mixture of sugar moieties and polyethylene glycol moieties.
Preferably the sugar moiety is or is derived from glucose or D-glucose.
D. Cationic lipid/NOI/packaging polypeptide complexes
[0071] A delivery vector/complex of the present invention is typically made by firstly contacting
a packaging polypeptide and an NOI in a sterile tube for about 10 mins at room temperature,
resulting in a condensed polypeptide/NOI complex. A common technique is to spot the
nucleic acid and protein alongside each other in the tube, but not in contact, and
initiate mixing by adding a few hundred microlitres of a liquid carrier, such as a
pharmaceutically acceptable carrier, excipient or diluent.
[0072] A further and preferred method of preparing a delivery vector/complex of the present
invention is by contacting a packaging polypeptide and an NOI during continuous vortexing.
[0073] Typically a ratio of NOI to polypeptide at least 1:1, preferably from 1:1 to 2:1,
more preferably from 1.4:1 to 1.9:1, more preferably from 1.5:1 to 1.8:1, is used.
We have found a ratio of NOI to polypeptide of approximately 1:0.6 (~ 1.7:1) to be
particularly effective. In some aspects, typically a ratio of polypeptide to NOI of
from 0.2 to 1.5, preferably from 0.3 to 1.2 (w/w), more preferably from 0.5 to 0.7
is used. In other embodiments the typically ratio of polypeptide to NOI is at least
10:1, or at least 20:1 (w/w). However, the optimum ratio may depend on the charge
: amino acid ratio of the packaging polypeptide. Generally, the lower the charge :
amino acid ratio, the higher the polypeptide : NOI ratio used.
[0074] Next, cationic lipids are added to the complex. The cationic lipids may, in one embodiment,
be part of a pre-formed liposome comprising two or more lipid constituents, such as
DC-Chol and DOPE. The cationic lipids are typically incubated with the polypeptide/NOI
complex for about 20 mins at room temperature. A further and preferred method of adding
the cationic lipids is in the form of a cationic liposome suspension. This final complex
may be stored at approximately -80°C with the addition of 10% sucrose (w/v) until
use.
[0075] The amount of liposome to NOI is typically in the order of from 3:1 to 20:1, preferably
from 6:1 to 15:1, more preferably from 8:1 to 14:1. We have found a ratio of liposome
to NOI of 12:1 to be particularly effective. In other embodiments the amount of liposome
to NOI is typically in the order of the 2:1 to 10:1, or from 3:1 to 6:1. Where cationic
lipids are used with neutral lipids, the ratio is typically in the order of 1:1.
[0076] In a highly preferred embodiment the ratio
|
liposome |
NOI |
polypeptide |
is |
3-20 |
1 |
0.5-1 |
preferably |
8-14 |
1 |
0.5-0.7 |
more preferably |
~12 |
1 |
~0.6 |
[0077] The delivery vector/complex is now ready for use. Although it is preferred to mix
the various components in the order described above, it is possible to combine the
components in any order. Where further polypeptide components are to be added, they
may be added at any stage but preferably together with the packaging polypeptide.
[0078] It may be desirable to include other components within the vectors/complexes, for
example ligands that bind to cell surface receptors, to provide the vectors/complexes
with a degree of selectivity for cell type. Ligands include peptides, glycoproteins,
oligosaccharides, lectins and antibodies and fragments thereof.
E. Administration
[0079] The delivery vector/complex of the invention is preferably combined with a pharmaceutically
acceptable carrier or diluent to produce a pharmaceutical composition (which may be
for human or animal use). Suitable carriers and diluents include isotonic saline solutions,
for example phosphate-buffered saline. The composition of the invention may be administered
by direct injection. The composition may be formulated for parenteral, intramuscular,
intravenous, subcutaneous, intraocular or transdermal administration or inhalation.
Typically, each NOI may be administered at a dose of from 10 ng to 10 µg/kg body weight,
preferably from 0.1 to 10 µg/kg, more preferably from 0.1 to 1 µg/kg body weight.
[0080] Alternatively, transfection of patient cells may be carried out
ex vivo by removal of patient tissue, transfection using a delivery vector/complex of the
invention, followed by reimplantation of the transfected tissue.
[0081] The routes of administration and dosages described are intended only as a guide since
a skilled practitioner will be able to determine readily the optimum route of administration
and dosage for any particular patient and condition.
F. Uses
[0082] The delivery vectors/complexes in the present invention may be used to efficiently
transfect eukaryotic cells, in particular mammalian cells, with NOIs. The delivery
vectors/complexes have been shown to be particularly efficient compared with prior
art compositions in transfecting neuronal cells. This has specific implications for
(i) research where neuronal cells are used and (ii) clinical applications where it
is desired to introduce NOIs into cells of the central of peripheral nervous system
of a human or animal. More generally, the delivery vectors/complexes in the present
invention may be used in a variety of NOI delivery applications such as gene therapy,
DNA vaccine delivery and
in vitro transfection studies.
[0083] Examples of diseases that may be targeted for treatment using the complexes/vectors
of the invention include diseases of the peripheral or central nervous system such
as neurodegenerative diseases and damage to nervous tissue as a result of injury/trauma
(including strokes). In particular, neurodegenerative diseases include motor neurone
disease, several inherited diseases, such as familial dysautonomia and infantile spinal
muscular atrophy, and late onset neurodegenerative diseases such as Parkinson's and
Alzheimer's diseases.
[0084] The delivery vectors/complexes of the invention may also be used to administer therapeutic
genes to a patient suffering from a malignancy. Examples of malignancies that may
be targeted for treatment include cancer of the breast, cervix, colon, rectum, endometrium,
kidney, lung, ovary, pancreas, prostate gland, skin, stomach, bladder, CNS, oesophagus,
head-or-neck, liver, testis, thymus or thyroid. Malignancies of blood cells, bone
marrow cells, B-lymphocytes, T-lymphocytes, lymphocytic progenitors or myeloid cell
progenitors may also be targeted for treatment.
[0085] The tumour may be a solid tumour or a non-solid tumour and may be a primary tumour
or a disseminated metastatic (secondary) tumour. Non-solid tumours include myeloma;
leukaemia (acute or chronic, lymphocytic or myelocytic) such as acute myeloblastic,
acute promyelocytic, acute myelomonocytic, acute monocytic, erythroleukaemia; and
lymphomas such as Hodgkin's, non-Hodgkin's and Burkitt's. Solid tumours include carcinoma,
colon carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, adenocarcinoma,
melanoma, basal or squamous cell carcinoma, mesothelioma, adenocarcinoma, neuroblastoma,
glioma, astrocytoma, medulloblastoma, retinoblastoma, sarcoma, osteosarcoma, rhabdomyosarcoma,
fibrosarcoma, osteogenic sarcoma, hepatoma, and seminoma.
[0086] Other diseases of interest include diseases caused by mutations, inherited or somatic,
in normal cellular genes, such as cystic fibrosis, thalessemias and the like.
[0087] Further areas of interest include the treatment of immune-related disorders such
as organ transplant rejection and autoimmune diseases. The spectrum of autoimmune
disorders ranges from organ specific diseases (such as thyroiditis, insulitis, multiple
sclerosis, iridocyclitis, uveitis, orchitis, hepatitis, Addison's disease, myasthenia
gravis) to systemic illnesses such as rheumatoid arthritis, and other rheumatic disorders,
or lupus erythematosus. Other disorders include immune hyperreactivity, such as allergic
reactions, in particular reaction associated with histamine production, and asthma.
[0088] The present invention will now be illustrated by means of the following examples
which are illustrative only and not limiting.
Description of the figures
[0089]
Figure 1 shows a plate;
Figure 2 shows a graph;
Figure 3 shows a plate;
Figure 4 shows a graph;
Figure 5 shows a graph;
Figure 6 shows a graph;
Figure 7 shows a graph;
Figure 8 shows a graph;
Figure 9 shows structures;
Figure 10 shows a graph;
Figure 11 shows a graph;
Figure 12 shows a graph;
Figure 13 shows a graph;
Figure 14 shows a graph;
Figure 15 shows a graph;
Figure 16 shows a plate;
Figure 17 shows a graph;
Figure 18 shows a graph;
Figure 19 shows a structure;
Figure 20 shows a reaction scheme;
Figure 21 shows a reaction scheme;
Figure 22 shows structures;
Figure 23 shows principle of miscellar incorporation;
Figure 24 shows a graph; and
Figure 25 shows a graph.
Detailed description of the figures 1 to 6
[0090]
Figure 1 - The Adenoviral core protein Mu1 is more efficient at binding plasmid DNA
than Polyomavirus core protein Vp1.
A) BSA has no effect on the electrophoretic mobility of pDNA. One microgram of pCMVβ
was incubated with 0 µg (lane 2), 5 µg (lane 3), 10 µg (lane 4), 15 µg (lane 5), 20
µg (lane 6), 25 µg (lane 7) and 30 µg (lane 8) of BSA for 10 minutes at room temperature
in 1X HBS. Samples were then analyzed on a 1% agarose gel for altered mobility. No
change in electrophoretic mobility by BSA was detected.
B) In contrast to BSA, Mu1 peptide dramatically interfered with the mobility of pDNA.
pCMVβ (1 µg) was incubated with 0.25 µg (lane 2), 0.5 µg (lane 3), 1 µg (lane 4),
2 µg (lane 4), 4 µg (lane 6), 6 µg (lane 7) and 0 µg (lane 8) recombinant Mu1 peptide
as in A. While ratios of protein to pDNA of 0.25 (w/w) (lane 2) did not alter migration
of the relaxed form of pCMVβ (upper band) a slight retardation of supercoiled pDNA
was seen (lower band). When ratios of 0.5 (w/w) or greater were used, however, migration
of both forms of pDNA was severely retarded.
C). The Polyomavirus protein Vp1 was much less efficient at preventing pDNA migration.
pCMVβ (1 µg) was incubated with 2 µg (lane 2), 4 µg (lane 3), 6 µg (lane 4), 8 µg
(lane5), 16 µg (lane 6), 32 µg (lane 7) and 0 µg (lane 8) Vp1. Only ratios of 6 or
higher (protein: pDNA, w/w) caused significant retardation of supercoiled pDNA (lane
6, lower band). Also, not until a ratio of 32 (w/w) was used was there any effect
on relaxed pDNA (lane 7, upper band). In all gels lane 1 corresponds to 1 Kb DNA marker
(BRL).
Figure 2 - β Galactosidase activity in ND7 cells transfected with pDNA-Mu1-cationic
liposome complexes.
ND7 cells were seeded at a density of 5 x 104 cells/well in 24 well culture dishes 24 hrs prior to transfection. Immediately prior
to transfection, cells were washed in serum-free media. Complexes were formed by incubating
pCMVβ with Mu1 prior to the addition of the cationic liposome DC-Chol/DOPE. In each
case 1 µg pCMVβ was complexed with 0.6, 6, 12, and 21 µg Mulpeptide. Each of these
combinations was then complexed with 3, 4 and 6 µg DC-Chol/DOPE. ND7 cells were exposed
to transfection complexes for 2 hours then maintained at 37°C, 5 % CO2 for another 24 hrs before being harvested and processed for β-galactosidase enzyme
assay. Numbers represent means ± SD, n=3.
Figure 3 - Mu1 enhances cationic liposome mediated transfection efficiency in the
neuronal cell line ND7.
ND7 neurons were plated in 24 well culture dishes at a density of 4X104 cells/well and allowed to grow for 24 hrs. The undifferentiated ND7 neurons were
then transfected with either pCMVb alone (A), pCMVb complexed with DC-Chol/DOPE (1/3,
w/w) (B) or with pCMVb complexed with Mu1 and DC-Chol/DOPE (1/12/6) (C). Forty-eight
hours later the cells were fixed and processed for histochemical detection of X-Gal.
As can be seen in panel C inclusion of Mu1 in the complex at an optimal ratio significantly
enhanced the number of X-Gal positive cells (blue).
Figure 4 - Mu1 is more efficient at enhancing cationic liposome mediated transfections
in ND7 cells than Vp1.
pCMVβ plasmid DNA was complexed to various amounts of polycationic peptide and then
mixed with cationic liposome at a ratio of 1:3 (pCMVβ: liposome; w/w). After being
washed briefly in serum free media, ND7 cells were exposed to the liposome-polycation-liposome
complexes for two hours and then returned to serum containing media. Twenty-four hours
later the cells were harvested and processed for β-galactosidase enzyme assay. Each
condition was performed in triplicate and each experiment replicated three times.
Numbers represent means ± SD.
Figure 5 - Mu1 enhances DC-ChoI/DOPE transfection in COS-7 cells.
COS cells were seeded at a density of 60-80% confluence in 24 well culture dishes
24 hrs prior to transfection. Immediately prior to transfection, cells were washed
in serum-free media. Incubating pCMVβ with Mu1 prior to the addition of the cationic
liposome DC-Chol/DOPE formed complexes capable of cellular transfection. In each case
1 µg pCMVβ was complexed with 12 µg Mulpeptide that had been found optimal for ND7
cells. The pCMVβ: Mu1 complexes were then mixed with 3, 4 and 6 µg DC-Chol/DOPE. COS
cells were exposed to transfection complexes for 2 hours then maintained at 37°C,
5% CO2 for another 24 hrs before being harvested and processed for β-galactosidase enzyme
assay. Numbers represent means ± SD, n=3.
Figure 6 - Transfection efficiency in differentiated ND7 cells with pCMVβ-Mulcationic
liposome complexes.
ND7 cells were plated in a 24 well culture plate at a density of 4X104 cells per well in normal growth media (+ serum). Twenty-four hours later the media
was replaced with differentiation media and the cells were grown for an additional
24 hrs. Three different differentiation medias were used; serum-free (-serum), normal
growth media plus 1 mM cAMP (cAMP), or reduced serum (0.5 %) plus 1 mM cAMP and 50
ng/ml nerve growth factor (NGF). The cells were then transfected with pCMVb complexed
with either DC-Chol/DOPE alone or Mu1 plus DC-Chol/DOPE. Forty-eight hours later the
cells were fixed and processed for X-Gal histochemistry and the percentage of positive
cells determined. In all cases the presence of Mu1 increased the number of positive
cells.
Interestingly the number of cells transfected was greater both with and without Mu1
for cells grown in cAMP.
EXAMPLES
Materials and Methods
Peptide Synthesis
[0091] Peptides Vp1 and Mu1 were synthesized on a Shimadzu PSSM-8 solid phase peptide synthesizer
using a five-fold excess of (9-fluorenyl)methoxycarbonyl (Fmoc)-protected L-amino
acids (Novabiochem) and the FastMoc™ reagents 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetra-methyluronium
hexafluorophosphate/hydroxybenzotriazole (HBTU/HOBt) (Advanced Chemtech Europe) as
the amide coupling agent. After resin cleavage and deprotection, desalting was performed
by gel filtration using a column of P2 Biogel (2 x 28 cm; Biorad) attached to an FPLC
system (Amersham Pharmacia Biotech UK) with 0.1% aqueous TFA as eluant at a flow rate
of 0.5-0.75 ml/min. Final preparative reverse-phase purification was achieved with
a Vydac column (C18, 5 µm, 2 x 25 cm; Hichrom) attached to a Gilson HPLC system (Anachem).
Peptides were eluted at 5ml/min by means of a linear gradient of acetonitrile in 0.1%
aqueous TFA and elution monitored at 220-230 nm.
[0092] The Vp1 peptide was prepared using a preloaded
L-Pro-2-chlorotrityl super acid labile resin (Novabiochem) (100 mg, 1.05 mmol/g, 0.1
mmol). Extended coupling times were used to incorporate all amino acid residues from
the sixth (Lys) through to the
N-terminal residue. After automated
N-terminal Fmoc deprotection with piperidine (20%, v/v) in dimethyl formamide, the
resin was isolated, washed with dimethylformamide (10 ml) and methanol (15 ml), and
then dried
in vacuo. Crude peptide was cleaved from the resin using ice cooled TFA (8 ml), containing
phenol (7%, w/v), ethanedithiol (2%, v/v), thioanisole (4%, v/v) and water (4%, v/v)
(known as Mixture A), and then precipitated with ice cold methyl-
tert-butylether (MTBE) (30ml). The subsequent pellet was then desalted and the crude peptide
mixture purified by reverse phase HPLC. After elution, fractions containing the desired
peptide (eluting with acetonitrile 68.5% v/v) were combined and lyophilized to give
the peptide as a white powder. Overall yield: 32 mg (15 µmol, 15%); MS (MALDI-TOF)
C
85H
151N
26O
26S
3: [
M + H]
+ calcd 2049.5, found 2050.2. The sequence was confirmed by amino acid composition
and sequence analysis. Homogeneity was judged >95% by HPLC analysis.
[0093] The Mu1 peptide was prepared using Gly-Wang resin (Novabiochem) (40 mg, 0.67 mmol/g,
0.03mmol). Normal coupling times were used throughout. After automated N-terminal
Fmoc deprotection as above, the resin was isolated and washed with dichloromethane
(20 ml) and methanol (20 ml) after which the resin was dried
in vacuo. Crude peptide was cleaved from the resin using Mixture A (8 ml) and precipitated
with MTBE (30 ml), all as above. Finally, the crude peptide mixture was desalted and
purified by reverse phase HPLC. After elution, fractions containing the desired peptide
(eluting with acetonitrile 17.2%) were combined and lyophilized to give the peptide
as a white powder. Overall yield: 65 mg (26 µmol, 80%); MS (ES) C
95H
170N
52O
21S
2: [
M+H]
+ calcd 2440.7, found 2440.6. Homogeneity was judged >95% by HPLC analysis.
DNA binding analysis
[0094] The purified peptides were reconstituted in sterile distilled H
2O at 3 mg/mL. Peptide and pDNA were complexed in 20 µL HEPES buffered saline (137
mM NaCl, 5 mM KCl, 0.75 mM Na
2HPO
4, 19 mM HEPES, pH 7.4) for 20 minutes at room temperature. Peptide: pDNA complexes
were subsequently analyzed by agarose gel electrophoresis (1%). Control incubations
for general macromolecular pDNA interactions were performed with varying amounts of
molecular biology grade purified bovine serum albumin (Sigma).
Cell Cultures
[0095] ND7s are a well-characterized cell line derived from the fusion of a neuroblastoma
(N18Tg2) with neonatal rat sensory neurons
28. The cell line was maintained in normal growth media (NGM) (Leibovitz's L-15 media
(BRL) enriched with 10 % Fetal bovine serum (BRL), 4 g/L glucose, 4 g/L sodium bicarbonate
(BRL), 100 IU/mL penicillin/streptomycin (BRL)) at 37°C and 5 % CO
2. The cells were plated onto 24 well plates (Costar) at a density that produced 70
% confluence after 24 hours.
[0096] Differentiation of ND7 cells was carried out using three previously described methods
28,
29. ND7 cells were seeded in NGM at a density of 4 X 10
4 cells per well in a 24 well culture dish (Nunc). Twenty four hours later the media
was replaced with either: a) NGM supplemented with 1mM adenosine 3':5'-cyclic monophosphate
(cAMP; Sigma), or b) serum-free differentiation media (50 % Hams F12, 50 % DMEM, 5
µg/mL Transierrin, 250 ng/mL Insulin, 0.3 µM sodium selenite), or c) low serum nerve
growth factor (NGF) media (L-15 supplemented with 2 mM glutamine, 4 g/L glucose, 4
g/L sodium bicarbonate, 10 u/mL penicillin, 10 g/mL streptomycin, 0.5% FCS, 1 mM cAMP,
50 ng/mL NGF (Alomone Labs)). Differentiated ND7 cells were grown in appropriate media
for 24 hrs at 37°C, 5 % CO
2 prior to transfection.
[0097] COS-7 cells (derived from Green Monkey kidney) were grown in RPMI 1640 media (BRL)
supplemented with 10% fetal bovine serum (BRL) and 100 IU/mL penicillin/streptomycin
(BRL).
Plasmid Constructs
[0098] All transfections utilized the reporter plasmid pCMVβ (Clontech, Palo Alto, CA) containing
the full-length sequence for
E.
coli β-galactosidase downstream of the human cytomegalovirus immediate-early promoter/enhancer
(Clontech). Stocks of plasmid DNA were prepared using standard molecular cloning techniques
and purified using the Qiagen Endotoxin-free plasmid purification system (Qiagen,
Dorking, UK).
Liposomes
[0099] DC-Chol/DOPE liposomes were prepared as previously described
30, 31. Briefly, 6 µmol of DC-Chol and 4 µmol of DOPE (supplied at 10mg/mL in CHCl
3) were added to freshly distilled CH
2Cl
2 (5 mL) under nitrogen. 5 mL of 20 mM Hepes (pH 7.8) was added to the mixture and
this was sonicated for 3 minutes. The organic solvents were removed under reduced
pressure and the resulting liposome suspension was then sonicated for a further 3
minutes. Liposome preparations were stored at 4°C.
Transfection Protocol
[0100] Since initial experiments determined that the presence of fetal bovine serum inhibited
transfection of ND7 cells serum-free differentiation media was used for all transfections.
Various amounts of DNA and liposome were placed in the bottom of a 7 mL sterile Bijou
container (Bibby Sterilin Ltd., Staffordshire, U.K.), but not in contact with each
other.
[0101] DNA and liposomes were combined by the addition of 400 µL serum-free differentiation
media and. gentle shaking. The DNA: liposome mixture was incubated at room temperature
for 20 to 30 minutes before being applied to the cells. The DNA/liposome mixture was
then applied to the cells and incubated at 37°C, 5% CO
2 for 2 hours after which this media was replaced with complete media. Twenty four
to 48 hours later the cells were fixed and processed for X-gal histochemistry as described
31 or harvested for β-galactosidase enzyme assays (Promega Corp.).
[0102] Cell counts were performed under x40 magnification using a Nikon Diaphot inverted
microscope. Each transfection was repeated at least three times and at least three
separate counts were made for each well.
[0103] Transfection complexes including the test peptides were generated in the following
manner. Various amounts of peptide was placed in the bottom of sterile polystyrene
containers alongside, but not in contact with 1 µg pCMVβ and mixed by adding 400 µl
serum free NGM media. The complexes were incubated at room temperature for 10 minutes
after which DC-Chol/DOPE was added. The pDNA/peptide/liposome complex was further
incubated at room temperature for 20 minutes and then administered to cells as above.
Example 1 - DNA Binding Analysis
[0104] Mu1 is a polycationic peptide comprised of 19 amino acids associated with the core
complex of Adenovirus (Table 1)
27,
32. We compared the DNA binding capacity of Mu1 with the mouse polyomavirus major capsid
protein Vp1 by interaction with plasmid DNA in a gel retardation assay. Vp1 is a 19
amino acid peptide that contains a nuclear localization signal
26 and contains fewer positively charged amino acids than Mu1. It was therefore predicted
to have a lower DNA binding capacity.
[0105] The NLS sequence in VP1 is underlined
[0106] Varying amounts of purified peptide were incubated at room temperature in HBS for
approximately 10 minutes and then analyzed by agarose gel electrophoresis. Without
the addition of peptide, supercoiled and relaxed circular plasmid DNA (pDNA) migrated
in the expected manner (Figure 1, lane 8).
[0107] Beginning at a DNA: Mu1 peptide ratio of 1:0.25 (w/w) the migration of plasmid DNA
was retarded (Figure 1). The migration of plasmid DNA was slightly affected at 1:0.25
ratios (w/w), but at a ratio 1:0.5 (w/w) the migration of plasmid DNA was severely
slowed and very little managed to migrate out of the wells. At ratios of 2:1 and above
pDNA was unable to migrate into the agarose gel and the ability of ethidium bromide
to interchelate into the plasmid was reduced. In contrast to Mu1, no effect on the
electrophoretic mobility of plasmid DNA was detected with Vp1 at pDNA: protein ratios
up to 1:8 (w/w) (Figure 1). The addition of 8 µg Vp1 to 1 µg plasmid DNA resulted
in a broadening of the supercoiled pDNA band. However, no effect was seen on the relaxed
pDNA band with Vp1 until a ratio of 1:32 (w/w) pDNA: protein was used. At this ratio,
both supercoiled and relaxed pDNA bands were significantly retarded and some DNA could
be seen retained in the well. No effect on electrophoretic mobility was detected when
pDNA was incubated with BSA at ratios up to 1:30 (w/w) pDNA: protein (Figure 1).
Example 2 - Transfection in undifferentiated ND7s
[0108] We examined the ability of Mu1 and Vp1 to enhance the transfection of a neuronal
cell line by cationic liposomes using a β-Galactosidase reporter gene assay. ND7 cells
were transfected with pCMVβ complexed to varying amounts of peptide and DC-Chol/DOPE.
We have previously shown that the cationic liposome DC-Chol/DOPE is capable of efficiently
transfecting the neuronally derived ND7 cell line
31. In this study we found that optimal efficiencies (>40%) were obtained in this neuronally
derived cell line using 1 µg plasmid DNA complexed with 3 µg DC-Chol/DOPE
31. Temporally, maximal levels of transgene expression are obtained between 48-60 hours
post transfection. Therefore, in order to maximize the chance of detecting improvements
in transfection we performed all our assays within 12-20 hours of transfection at
a time when levels of reporter gene expression were lower. Previously we found a pDNA:
liposome ratio of 1:3 (w/w) optimal for transfections in ND7 cells
31. We therefore compared the effect of various amounts of peptide on transfections
at ratios of 1:3, 1:4 and 1:6 pDNA: DC-Chol/DOPE. The gel retardation analysis suggested
that an approximate ratio of 1:0.5 (w/w), pDNA: Mu1, was enough to essentially bind
all of the plasmid DNA (Figure 1). However, initial experiments using this ratio and
liposomes did not affect transfection efficiencies (not shown). The volumes used to
generate transfection complexes were much larger than those used to perform the gel
retardation assay (400 µL vs. 20 µL). Therefore we tested larger quantities of Mu1
that were of a similar concentration in solution as that used in the gel retardation
assay. We compared the effect 0.6, 6, 12 and 21 µg of Mu1 peptide would have on DC-Chol/DOPE
mediated transfections. We found that Mu1 was able to improve cationic liposome mediated
transfection efficiencies over 4-fold. The greatest improvement in transfection efficiencies
occurred when the relative ratios of 1/12/6, pCMVβ/Mu1/(DC-Chol/DOPE) (w/w/w) were
used. This combination led to an 11-fold increase in transfections compared to DNA
alone (Figure 2).
[0109] The β-galactosidase reporter gene assay provides a measure of the overall level of
β-galactosidase produced, but gives no information regarding the number of cells transfected.
For this reason, we also performed cell counts on transfected ND7 cells. Cells were
seeded at a density of 4 x 10
4 in 24 well culture plates. After 24 hours the cells were washed briefly in serum-free
media and transfected with pCMVβ complexed to DC-Chol/DOPE and Mu1 peptide. The ratios
used were those found to be optimal in the reporter gene assay, 1:12:6, pCMVβ:Mu1
:DC-Chol/DOPE. Using these ratios, we found a 6-fold increase in the number of β galactosidase
positive cells (Figures 3 & 6). No obvious cell loss was detected with the Mu1 complex
at any of the concentrations utilized. Similarly the concentration of protein in the
cellular lysates used for β-Galactosidase reporter gene assay did not significantly
vary with untransfected cells (data not shown).
[0110] In contrast, no improvement on transfection efficiency could be found with Vp1 (Figure
4). No improvement in transfection efficiencies over naked DNA was seen with pCMVβ
complexed to Mu1 alone.
[0111] In order to see whether improved transfections could be achieved in other cell types
we performed a similar analysis on COS-7 cells. Mu1 also improved liposome-mediated
transfection in COS-7 cells (Figure 5). The same ratio of pDNA: Mu1: liposome optimal
for ND7 cells was best for COS-7 cells. A similar degree of improvement was also seen
(3.7 fold) over cationic liposomes alone.
Example 3 - Transfection in differentiated ND7s
[0112] We also examined the ability of Mu1 to improved cationic liposome-mediated transfection
in differentiated ND7s. The ND7 cell line is derived from a fusion of primary rat
dorsal root ganglia (DRG) neurons and the mouse neuroblastoma N18Tg2
28. ND7 cells can be differentiated in a variety of manners including the withdrawal
of serum, cAMP administration or exposure to reduced serum plus cAMP and nerve growth
factor. Differentiation of ND7s leads to the expression of cellular properties associated
with their parental nociceptive sensory neurons including a reduction in cell division
and the onset of neurite outgrowth. ND7 cells were seeded in 24 well culture plates
and 24 hours later differentiated. Fifteen to 20 hours following the onset of differentiation,
they were transfected as above. Fifteen to 20 hours following transfection, cells
were fixed and processed for X-gal histochemistry. Consistent with previous observations,
transfection efficiencies varied greatly between the three differentiated groups.
ND7 cells differentiated by withdrawal of serum exhibited the lowest levels of transfection
(1.3%) while highest levels were seen in the cAMP group (8%) and intermediate levels
in the low serum/cAMP/NGF group (4.7%) (Figure 5). In all three conditions, however,
inclusion of Mu1 polypeptide in the transfection complex improved the transduction
of differentiated ND7 cells. ND7s differentiated by either cAMP alone or exposure
to low serum/cAMP/NGF exhibited increased efficiencies of greater than 6 fold (Figure
5). The greatest improvement in efficiencies was seen, however, in the group differentiated
by serum withdrawal. Here, increases of greater than 10 fold were observed.
Complexes of Mu1 peptide and DNA
[0113] As shown in Example 1 (DNA binding Analysis) using gel electrophoresis, the migration
of plasmid DNA was severely retarded and little DNA migrated out of the wells above
a Mu1:DNA 0.5:1.0 (w/w). This implied that Mu1 peptide was strongly interacting with
DNA and might neutralise and condense nucleic acids to form small particles suitable
for gene delivery. The size of Mu1:DNA (MD) particle sizes were examined over the
Mu1: DNA ratio range indicated in Fig.7.
[0114] MD particles were prepared by mixing. Briefly, appropriate aliquots of Mu1 peptide
in deionized water were added to plasmid DNA (pCMVβ) (final concentration 220µg/ml)
in 20 mM Hepes buffer, pH7.0. After mixing well, each mixture was incubated for 10
min at 20°C. Immediately after incubation each mixture was diluted with the Hepes
buffer (final DNA concentration 24µg/ml) and subjected to particle size analysis by
photon correlation spectroscopy (N4 plus, Coulter). All measurements were performed
at 20°C and data collected at an angle of 90°. Unimordal analysis was used to calculate
the mean particle size and standard distribution (S.D.).
[0115] Interestingly, though Mu1 bound DNA and formed complexes over the complete range
examined, the particle size varied considerably in response to the Mu1:DNA ratio.
Stable, small nano-particles were formed within the Mu1:DNA ratio 0.3 to 1.2 (range
L) and over 5 (range H). Intermediate ratios resulted in heavy aggregation with the
size of complex particles growing over the time of incubation to reach more than 2µm
in size (Fig .7).
Example 4 - Preparation of LMD in the range L
[0116] We determined whether liposome-Mu1-DNA complexes with a low MD:DNA ratio could form
stable nano-particles and whether the resulting complex particles could have good
transfection activities.
[0117] Preparation of liposomes; DC-Chol (30µmol) and DOPE (20µmol) were combined in dichloromethane.
The organic solvent was removed under reduced pressure using a rotary evaporator and
the residue dried for 3h
in vacuo. Following this, 4mM Hepes buffer, pH7.0 (3ml), was added to the lipid film with vortex
mixing. After brief sonication (2-3min), the resulting cationic liposome suspension
was extruded by means of an Extruder device (Lipex Biomembranes) three times through,
two stacked polycarbonate filters (0.2µm Millipore) and then ten times through two
stacked polycarbonate filters (0.1µm Millipore) to form small liposomes (109nm average
diameter by PCS) (approx. 8-10 mg/ml depending upon the preparation).
[0118] Preparation of Liposome:Mu1 :DNA (LMD) complexes; Mu1 peptide (0.12mg in deionised
water, peptide concentration 3.5mg/ml) was added to a solution of plasmid DNA (pCF1-CAT)
(0.2mg, plasmid concentration typically 1.0mg/ml) in 4mM Hepes buffer during continuous
vortexing. Cationic liposome suspension (total lipid 2.4mg, 4 µmol) was then introduced
resulting in the formation of small particles with narrow size distribution (168nm±58nm)
as measured by PCS. This LMD (final DNA concentration 0.14mg/ml) was stored in -80°C
with the addition of 10% sucrose (w/v) until use. No particle size deviation was observed
over one month.
[0119] Liposome:DNA (LD) complexes (lipoplexes) were prepared for control experiments with
a Liposome:DNA ratio of 3:1 (w/w), the optimal composition for transfection ofND7
cells.
[0120] Transfection in ND7 cells; ND7 cells were seeded in normal growth medium (NGM) (with
10% serum) at a density of approximately 4x10
4 cells per well, in a 24-well culture plate. After 24h, cells were washed by brief
exposure to NGM (serum free) and then treated with solutions containing LMD or LD
complexes, prediluted with NGM (serum free) (final DNA concentration 3.2µg/ml in all
cases), for the time periods indicated. Cells were then washed again and incubated
for a further 48h prior to harvesting. Levels of transfection were determined by chloramphenicol
transferase (CAT) enzyme assay using
14C-CAM as substrate (Promega). Transfection activity was expressed as a percentage
(%) conversion of the imputed
14C-CAM by the enzyme.
[0121] We found much higher reporter gene expression with LMD compared to LD mediated transfection.
In fact, LMD transfection resulted in 16 times more CAT enzyme activity after a transfection
time of 10 mins, and 6 times as much after a transfection time of 60 min compared
with LD-mediated transfection (Fig.8). Significant transfection was observed with
LMD even when the transfection time was as short as 10 min. This data illustrates
how rapidly LMD particles are able to enter cells.
Example 5 - Cationic lipid (cytofectin) variations
[0122] We determined whether LMD complexes could be bettered by incorporating poly cationic
cholesterol lipids (WO 97/45442). CDAN (B198), ACHx (CJE52) and CTAP (B232) (Fig.
9) were used to make cationic liposomes in place of DC-Chol. Each cationic liposome
system used was composed of 60mol% of cationic lipid and 40mol% of DOPE and prepared
as described in Example 4. The following different LMD complex systems were prepared
and compared: LMD(DC-Chol), LMD(B198), LMD(CJE52), and LMD(B232). All LMD systems
were prepared with cationic liposomes (total lipid 20µmol) and 0.6mg of Mu1 peptide
per 1.0mg of DNA (pCMVβ), as described above. Particles were shown to be under 200nm
in diameter.
[0123] Liposome:DNA (LD) complex mixtures (lipoplexes) were prepared for control experiments
with a Liposome:DNA ratio of 3:1 (w/w), the optimal composition for transfection of
ND7 cells.
[0124] Transfection in ND7 cells; ND7 cells were seeded in NGM (with 10% serum) at a density
of approximately 4x10
4 cells per well, in a 24-well culture plate. After 24h, cells were washed by brief
exposure to NGM (serum free) and then treated with solutions containing LMD or LD
complexes, prediluted with NGM (serum free) (final DNA concentration 2.5µg/ml in all
cases), for 1h. Cells were then washed again and incubated for a further 48h prior
to processing for histochemical staining with X-gal. The number of cells stained blue
were counted under an inverted microscope.
[0125] In all cases LMD formulations worked better than corresponding LD systems prepared
with the same poly cationic cholesterol lipids (Fig. 10). The rank order in transfection
efficiency was LMD(B198) > LMD(DC-Chol) > LMD(CJE52) >> LMD(B232). The same rank order,
B198 > DC-Chol > B232, was observed with corresponding LD systems.
Example 6 - Amount of Mu1 peptide in the range L
[0126] We examined the effect of Mu1: DNA ratio (at the range L in Fig.7) on transfection
activity.
[0127] Cationic liposomes composed of cationic lipid B198 and DOPE (3:2 m/m) were prepared
as the same manner described in Example 4. A series of MD complex mixtures (Mu1:DNA
ratio varying from 0.3 to 1.2) were prepared and complexed with the cationic liposome.
The resulting LMD systems were comprised of liposome:Mu1 :DNA (pCMVβ) in ratios of
12:0.3:1, 12:0.6:0.6, 12:0.9:1 and 12:1.2:1 w/w/w respectively. Measured sizes of
LMD particles were approximately 150nm.
[0128] Transfection activities were evaluated in vitro using Panc-1 cells (human pancreatic
cancer cell line). The cells were seeded at an approximate density of 5x10
4 per well in a 24-well culture plate in RPMI supplemented with 10% FCS and grown for
24h in the presence of 5% CO
2 at 37°C. Cells were washed by brief exposure to RPMI and then treated with solutions
of LMD complexes, prediluted with RPMI (final DNA concentration 5.0µg/ml in all cases),
for 30min. Cells were then washed again and incubated for a further 48h in RPMI supplemented
with 10% FCS prior to harvesting and the assay of β-galactosidase enzyme activity
using a standard assay kit (Promega).
[0129] As shown in Fig.11, the optimum liposome:Mu1:DNA ratio for transfection of Panc-1
cells was found to be 12:0.6:0.6. Otherwise, excellent transfection results were obtained
with these low ratio Mu1 LMD complexes.
Example 7 - Amount and composition of lipids
[0130] To investigate the effect of varying the ratio of cationic lipid to DOPE as well
as the ratio of total lipid to Mu1 and DNA, a series of LMD systems were prepared
using B198 as the preferred cationic lipid. Cationic liposomes composed of 60mol%
of B198 and 40mol% of DOPE (3:2 m/m), 50mol% of B198 and 50mol% of DOPE (1:1 m/m)
and 33mol% of B198 and 67mol% of DOPE (1:2 m/m) were prepared and combined with a
standard MD complex mixture (Mu1:DNA 0.6:1 w/w) at ratios indicated in Fig.12, according
to the method in Example 4.
[0131] Liposome:DNA (LD) complex mixtures (lipoplexes) were prepared for control experiments
with a Liposome:DNA ratio of 3:1 (w/w). All LMD systems were found to have a larger
average size when lower amounts of cationic liposomes were'complexed with MD complexes.
However, the size of LMD particles composed of more than 12µmol lipids/mg DNA remained
less than 200nm, whilst that of 12 to 6µmol lipids/mg DNA climbed above that value.
Occasionally, visible aggregation was observed during the preparation of LMD systems
comprised of 6µmol lipids/mg DNA.
[0132] Transfection activities were determined with Panc-1 cells (Fig.12). The cells were
seeded at an approximate density of 5x10
4 per well in a 24-well culture plate in DMEM supplemented with 10% FCS and grown for
24h in the presence of 5% CO
2 at 37°C. Cells were washed by brief exposure to DMEM and then treated with solutions
containing LMD or LD complexes, prediluted with DMEM (final DNA concentration 5.0µg/ml
in all cases), for 2h. Cells were then washed again and incubated for a further 48h
in DMEM supplemented with 10% FCS prior to harvesting and assay of β-galactosidase
enzyme activity using a standard assay kit (Promega).
[0133] The maximum transfection activity was not significantly different with the three
cationic different lipid to DOPE formulations tested. In the case of LMD systems prepared
with B198:DOPE(1:2 m/m) the maximum transfection was achieved at a liposome:DNA ratio
of around 12.5µmol lipid /mg DNA. The transfection activities of LMD systems prepared
with B198:DOPE (3:2 m/m) and B198:DOPE (2:2 m/m) cationic liposomes reached a plateau
at liposome:DNA ratios greater than 12.5µmol lipid/mg DNA. All LMD systems analysed
tended to show low transfection activities at low liposome:DNA ratios (Fig. 12). It
is considered that LMD formulations composed of low amounts of Mu1 peptide should
need larger amounts of cationic liposomes compared to the formulations prepared with
higher amounts of Mu1 peptide in order for the respective LMD systems to show full
transfection activity.
Example 8 - Comparison of Mu1 peptide with protamine
[0134] Protamine is a naturally occurring cationic peptide abundant in piscine sperm and
is potent in neutralising and condensing DNA. The transfection activity of protamine
was compared with that of Mu1 peptide. Mu1 peptide or protamine sulfate (Sigma, grade
X from Salmon) was complexed with DNA (pCMVβ) and then cationic liposomes (B198:DOPE
in a ratio of 3:2 m/m) giving a liposome:peptide:DNA ratio of 12:0.6:1 (w/w/w).
[0135] The transfection activities were examined in Swiss 3T3 cells. The cells were seeded
at an approximate density of 2x10
4 per well in a 24-well culture plate in DMEM supplemented with 10% FCS and grown for
48h to complete confluence in the presence of 5% CO
2 at 37°C. Cells were washed by brief exposure to DMEM and then treated with solutions
containing LMD or LD complexes, prediluted with DMEM (final DNA concentration 50µg/ml
in all cases), for 1 or 2h. Cells were then washed again and incubated for a further
48h in DMEM supplemented with 10% FCS prior to harvesting. The level of β-galactosidase
enzyme activity was determined with a standard assay kit (Promega).
[0136] As shown in Fig. 13 the complexes comprising Mu1 peptide showed better transfection
of these confluent cells than those comprising protamine.
Example 9 - Alternative cationic peptides
[0137] In order to examine the effects of various alternative cationic peptides on transfection
activities, a series of liposome:cationic peptide:DNA complexes were prepared and
their relative transfection abilities analysed
in vitro. The peptides used were poly-lysine hydrochloride (average molecular weight 3970,
Sigma), poly arginine hydrochloride (average molecular weight 11800, Sigma) a peptide
derived from protein V, pV (p5, sequence shown below), a peptide analogue of Mu1 (V,
sequence shown below) and Mu1 peptide itself. The p5 peptide and V peptide were synthesized
using the same solid-phase peptide synthesis methodology as was used to prepare Mu1
peptide.
[0138] Each peptide was combined with cationic liposome (DC-Chol:DOPE 3:2 m/m) and DNA (pCMVβ)
in the liposome:peptide:DNA ratio of 12:0.6:1 (w/w/w) as described in Example 4. The
transfection activities were examined using HeLa cells (human epithelial cells). The
cells were seeded at an approximate density of 5x10
4 per well in a 24-well culture plate in DMEM supplemented with 10% FCS and grown for
24h in the presence of 5% CO
2 at 37°C. Cells were washed by brief exposure to DMEM and then treated with solutions
containing LMD or LD complexes, prediluted with OPTIMEM (Gibco) (final DNA concentration
1.0µg/ml in all cases), for 30min. Cells were then washed again and incubated for
a further 48h in DMEM supplemented with 10% FCS prior to harvesting. The level of
β-galactosidase enzyme activity was determined with a standard assay kit (chemiluminescent,
Roche).
[0139] As shown in Fig. 14, the cationic peptides derived from adenovirus (Mu1 and p5) and
the Mu1 analogue (V) revealed excellent transfection activity compared to complexes
prepared using the synthetic cationic polypeptides, poly lysine and poly arginine.
Example 10 - Comparison with Transfast using Panc-1
[0141] LMD and LD were prepared by the same method described in Example 4 except for use
of pCMVβ. Transfast (Promega) DNA complex was prepared according to manufacturer's
protocol.
[0142] Transfection activities were evaluated in vitro using Panc-1 cells. The cells were
seeded at an approximate density of 5x10
4 per well in a 24-well culture plate in RPMI supplemented with 10% FCS and grown for
24h in the presence of 5% CO
2 at 37°C. Cells were washed by brief exposure to RPMI and then treated with solutions
containing LMD or LD complexes, prediluted with RPMI (final DNA concentration 5.0µg/ml
in all cases), for the times indicated. Cells were then washed again and incubated
for a further 48h in RPMI supplemented with 10% FCS prior to harvesting. The level
of β-galactosidase enzyme activity was determined with a standard assay kit (Promega).
Transfection with Transfast:DNA complex was performed in serum free medium (optimum
conditions) for 1h.
[0143] As shown in Fig. 15, LMD showed better transfection activity than the Transfast:DNA
complex and LD. These results are completely consistent with those found with ND-7
cells.
Example 11 - Comparison with Lipofectamine using human bronchial cells
[0144] The transfection activity of LMD complexes was compared with that of Lipofectamine
(Gibco) complexed with DNA using HBE cells (human bronchial epithelium cell).
[0145] The cells were seeded in a 12-well culture plate in DMEM supplemented with 10% FCS
and grown for 24h in the presence of 5% CO
2 at 37°C. Cells were washed by brief exposure to DMEM and then treated with solutions
containing either LMD (prepared as in Example 4) or LD (prepared from lipofectamine:DNA
12:1 w/w) complexes, prediluted with OPTIMEM (Gibco) (final DNA concentration 5.0µg/ml
in all cases), for the indicated times (see Fig 16). Cells were then washed again
and incubated for a further 48h in DMEM supplemented with 10% FCS prior to processing
for histochemical staining with X-gal.
[0146] LMD showed a better transfection activity than lipofectamine (Fig. 16) and exhibited
a more rapid uptake by HBE cells. Similar results were seen with ND7 and Panc-1 cells.
Example 12 - Comparison with LT1 using rat brain; ex vivo experiment
[0147] We assessed transfection activities in organotypic cultures from the rat brain using
a reporter DNA (pCMVβ) in order to mimic an in vivo model. Brain slices were maintained
on transparent porous membranes and were observed to maintain their intrinsic connectivity
and cytoarchitecture to a large degree.
[0148] LMD and LD were prepared as shown in Example 4. LT1 is a polyamine transfection reagent
manufactured by PanVera Co. A complex containing cationic liposome (DC-Chol:DOPE,
3:2 m/m), LT-1 and pCMVβ plasmid in the ratio 3:3.2:1 (w/w/w) was prepared. Brain
slices were treated with solutions containing LMD, LD or liposome:LT1:DNA for 2h (Murray
et al., Gene Ther. 1999,
6, 190-197). In all cases no morphological changes in the sections were observed during
the experiment. After 48h incubation post-transfection, cells were harvested, X-gal
stained and the number of blue cells counted on a slice (Fig.17).
[0149] At a DNA dose of 5.0µg (2ml culture), LMD gave an apparently larger number of blue
stained cells than LD or LT1 complex after X-gal staining. At a dose as low as 129ng,
LMD showed considerable transfection activity, still higher than that of LD (DC-Chol:DOPE
complexed to DNA, 3:1 w/w ratio) (DNA dose 5.0µg). We found much higher reporter gene
expression with LMD compared to transfection mediated by LD and liposome:LT1:DNA complexes.
In fact, LMD mediated transfection was over 19 times more effective than LD and over
4 times more effective than liposome:LT1:DNA at comparable doses.
Example 13 - Comparison with GL-67 cationic liposomes; in vivo experiment in mouse
lung
[0150] We assessed the transfection activity in mouse lung in vivo of LMD (prepared as described
in Example 4 using DC-Chol:DOPE cationic liposomes [3:2, m/m] and pCF1-CAT plasmid),
comparing this with the transfection activity of cationic liposomes GL-67:DOPE:DMPE-PEG
5000 (1:2:0.05 m/m/m) complexed with pCF1-CAT plasmid (LD) (liposome:DNA ratio 5.4:1 w/w)
used to great effect in lung clinical trials (Alton
et al., Lancet, 1999,
353, 947-954).
[0151] LMD (final DNA concentration 0.14mg/ml; 100µl volume; DNA dose 14µg) was instilled
into the lungs of Balb/c mice. GL-67:DOPE:DMPE-PEG
5000 (1:2:0.05 m/m/m) was complexed with pCF1-CAT plasmid (final DNA concentration 0.8mg/ml;
100µl volume; DNA dose 80µg) and this LD complex was similarly instilled into the
lungs of Balb/c mice. After 48h, the lungs were homogenised and assayed for CAT activity.
Error bars indicate s.e.m.
[0152] The results show (Fig. 18) that LMD and the GL-67 containing LD system gave essentially
equivalent levels of transfection in vivo even though the LMD system was delivering
a five fold lower DNA dose.
Example 14 - Sugar modified LMD systems
[0153] Unspecific interactions of LMD with the biological environment should be minimised
for
in vivo applications. For example, during intravenous administrations undesired interactions
with blood components (salts, proteins...) and non-target cells are important obstacles.
This opsonization of foreign particles with plasma proteins presents one of the first
steps in the natural process of removal of foreign particles by the innate immune
system. To reduce proteins binding and salt induced aggregations, naturally occurring
polysaccharides can be coupled to LMD. This carbohydrate modification of LMD can be
as well applied for targeting of LMD to carbohydrates receptors.
[0154] To obtain the desired effect, we designed the neoglycolipids described in Fig. 19.
Those compounds are based onto three distinct domains.
ACHx (CJE 52): This lipid (see Fig. 9) was chosen as generic lipid platform for the desired neoglycolipids.
The cholesterol aliphatic ring system represents a very hydrophobic area that inserts
inside the lipid coat of LMD or LD particles acting as a neoglycolipid anchor.
Carbohydrate motif: The choice of oligosaccharides was limited by the complexity of any chemistry involving
carbohydrate modifications. We decided to use the long chain commercially available
carbohydrates maltotetraose and maltohepataose as proof of principle.
Linker: Use of a chemoselective linkage proved efficient and flexible, allowing us to synthesise
a wide range of neoglycolipids. This chemoselective technique was based upon a conversion
of CJE52 into an hydroxylamino lipid that was able to couple directly to unprotected
carbohydrates. The synthesis of a typical hydroxylamino-CJE52 is shown in Figure 20
- Scheme 1 and the coupling of the carbohydrate moiety onto the linker is based on
the glycosylation of an O-substituted hydroxylamine (The principle of the reaction
with Glucose is illustrated in Figure 21 - Scheme 2). Following this strategy, Maltotetraose
and Maltoheptaose were coupled to obtain GLU4 and GLU7 compounds (Structure in Fig.
22).
[0155] The glyco-modification of LMD was based on the natural ability of neoglycolipid micelles
to dissociate and free lipids incorporate into LMD membranes. Firstly LMD were formulated
from DC-Chol:DOPE cationic liposomes, Mu1 peptide and pCMVβ plasmid as described in
Example 4. Thereafter, a suspension of neoglycolipid micelles in Hepes Buffer, pH
7.0 was added to LMD mixtures and the whole incubated for 30 min at 20°C before storage
at -80°C (Fig. 23).
[0156] Neoglycolipids Stabilisation of LMD: the stabilisation effect of neoglycolipid modified
LMD was evaluated by incorporation of 7.5mol% of GLU4 or GLU7 into LMD. The lipid
layer of LD systems is known to aggregate after salt exposure. Therefore, the sizes
of LD (final DNA concentration 1µg/ml) particles were evaluated after 30min at 37°C
in OPTIMEM by Photon Correlation Spectroscopy (N4 plus, Coulter). Unimodal analysis
was used to evaluate the mean particle size. The average percentage increase in LD
particle size is shown (Fig. 24). The same procedure was followed for the basic LMD
system, LMD(GLU4) and LMD(GLU7) (final DNA concentrations 1µg/ml).
[0157] The results indicate that LMD is more stable than LD in solution but also show that
the presence of GLU4 and GLU7 has an enhanced anti-aggregation stabilising effect
on LMD particles at 7.5mol%.
[0158] In vitro transfection efficiency: transfection activity was determined with Hela
cells seeded at 5x10
4 cells per well in 24-well culture plates and grown to approximately 70% confluence
in DMEM supplemented with FCS at 37°C and in the presence of 5% CO
2. Cells were washed in PBS and then treated with solutions containing LMD complexes,
prediluted with DMEM containing FCS at the indicated percentages (%) (final DNA concentration
5.0µg/ml in all cases), for 30min. Cells were further washed and then incubated for
a further 48h in normal medium (NGM) prior to harvesting. The level of β-galactosidase
expression was determined with a standard assay kit (chemiluminescent, Roche).
[0159] The results indicate an enhancement of the transfection efficiency due to Sugar modification
in both 0% and 50% Serum conditions (Fig. 25).
Discussion
[0160] We have previously shown that DC-Chol/DOPE liposomes are efficient at transfecting
the neuronally derived ND7 cell line
31. DC-Chol has been used successfully outside the CNS in a variety of tissues and has
undergone clinical trials for gene therapy treatments of cystic fibrosis
33,
34. Also, DC-Chol liposomes have been shown not to exhibit cytotoxic side effects
35,
36. For these reasons we wish to develop improved formulations of these liposomes for
use in neural cells.
[0161] We describe here the use of a virus-coded protein for cellular transfection. We found
that Mu1, when used in combination with the cationic liposome DC-Chol/DOPE was able
to improve significantly cellular transfection. This effect was most likely due to
the ability of Mu1 to condense pDNA and could be optimized by varying the ratios of
polypeptide, pDNA and cationic liposome. Significantly, the enhancement in transfection
efficiency was more pronounced on differentiated cells. As mentioned above, ND7 cells
were derived from primary DRGs. Differentiating ND7 cells induces a phenotype similar
to their parental peripheral sensory neurons including the induction of neurite outgrowth,
a reduction in overall proliferation and a reduction in transfectability
28, 37. An enhancement in transfection efficiency in differentiated ND7s may reflect an
enhanced ability to promote transfections in primary neurons or
in vivo.
[0162] The success of non-viral gene delivery vehicles as viable alternatives to virus vector-based
systems is dependent on the development of complexes with higher and longer lasting
transfection efficiencies. Since the initial identification of cationic liposomes
as vehicles for the transfer of genetic material into cells there has been a large
push to develop better cationic liposome formulations
5,
7. Most attempts at improving cationic liposomes have been based on structural modifications
to the molecule itself
30. Novel formulations have been developed which have improved transfection efficiencies
30. However, particular cell types behave differently in regards to cationic liposomal
transfection. For example, we found the polypeptide Mu1 better at enhancing cationic
liposome mediated transfection than Vp1. This was probably due to Mul's greater charge
ratio. While both peptides are approximately the same molecular weight, the overall
charge ratio of Mu1 was more than twice that of Vp1 (Table 1). Consistent with this
Mu1 was able to retard the electrophoretic mobility of plasmid DNA at less than 1/60
th the concentration demonstrating how tightly Mu1 is able to bind DNA. While a small
shift in pDNA mobility was detected when 0.25 µg Mu1 was complexed to 1 µg pCMVβ,
almost all of the plasmid was retained near the loading well following addition of
0.5 µg Mu1 (Figure 1). A 0.5/1.0 (w/w) ratio of Mu1 to pCMVβ corresponds to a 1000/1
molar ratio. Each molecule of Mu1 contains 12 residues that could potentially carry
a positive charge. The theoretical charge ratio of Mu1 to pCMVβ would then be 1.6
(12000 Mu1 cations to 7500 pCMVβ anions). This ratio should completely neutralize
the negative charges on pCMVβ thus completely retarding its migration as seen.
[0163] A direct comparison between the amount of Mu1 that significantly retarded plasmid
DNA migration and that which optimally enhanced transfections could not be made since
the method of preparation was different. The peptide-pDNA-liposome transfection complexes
were prepared in larger volumes (see Materials and Methods). Although it took 24 times
as much Mu1 (12 µg /1 µg pCMVβ) to achieve optimal enhancement of transfection efficiencies
as it did to retard migration in an agarose gel, the concentration in solution was
similar (25ng/mL, pDNA retardation; 30ng/mL, optimal transfections). The presence
of Mu1 also altered cationic liposome pDNA interactions. The optimal ratio of DC-Chol/DOPE
to pCMVβ in the presence of Mu1 was 6/1, twice that previously found optimal in neuronal
cells
31,
38. Theoretically the amount of Mu1 used should have completely neutralized the positive
charges on pCMVβ, which would have prevented further complexing with DC-Chol/DOPE.
Clearly this was not the case since much improved transfection efficiencies were attainable.
It's likely that not all the possible charged amino acids were protonated in our buffer
conditions. Why more cationic liposomes are required to improve transfections is not
clear and we are currently working to address this question.
[0164] Finally a point should be made regarding the nuclear localization signal embedded
within Vp1. Recent evidence in our laboratory (unpublished observations) and in others
10, 11,
39, 40 has suggested that nuclear transport of transfected material may be inefficient in
lipofection. For this reason attempts have been made to pre-condense DNA with polycations
containing peptide sequences known to have nuclear localizing capabilities with the
aim of improving nuclear uptake of transfected DNA
17, 20, 22. We found however, that the more efficient DNA condensing properties of Mu1 far outweighed
the nuclear localizing capacity of Vp1 in terms of improving transfection efficiencies.
Similarly Fritz et al.,
22 found no difference in transfection efficiencies between recombinant human histone
(H1) and a modified version containing the SV40 large T antigen nuclear localizing
sequence. Other studies have suggested that the presence of an NLS does improve nuclear
accumulation of transfected pDNA albeit via specific intracellular pathways
41, 42.
[0165] All publications mentioned in the above specification are herein incorporated by
reference. Various modifications and variations of the described methods and system
of the invention will be apparent to those skilled in the art without departing from
the scope and spirit of the invention. Although the invention has been described in
connection with specific preferred embodiments, it should be understood that the invention
as claimed should not be unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention which are obvious
to those skilled in molecular biology or related fields are intended to be within
the scope of the following claims.
References
[0166]
1. Wood MJA et al. Inflammatory effects of gene-transfer into the CNS with defective HSV-1 vectors.
Gene Ther 1994; 1: 283-291.
2. Byrnes AP et al. Adenovirus gene-transfer causes inflammation in the brain. Neuroscience 1995; 66: 1015-1024.
3. Naldini L et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral
vector [see comments]. Science 1996; 272: 263-267.
4. Miller AD. Cationic liposomes for gene delivery. Angewandte Chemie-International Edition 1998; 37: 1769-1785.
5. Lee RJ, Huang L. Lipidic vector systems for gene transfer. Critical Reviews in Therapeutic Drug Carrier Systems 1997; 14: 173-206.
6. Gao X, Huang L. Cationic liposome-mediated gene-transfer. Gene Ther 1995; 2: 710-722.
7. Felgner PL et al. Lipofection - a Highly Efficient, Lipid-Mediated Dna-Transfection Procedure. Proceedings Of the National Academy Of Sciences Of the United States Of America 1987; 84: 7413-7417.
8. Farhood H, Serbina N, Huang L. The role of dioleoyl phosphatidylethanolamine in
cationic liposome mediated gene transfer. Biochim Biophys Acta 1995; 1235: 289-295.
9. Caplen NJ et al. In-vitro liposome-mediated DNA transfection of epithelial-cell lines using the cationic
liposome DC-Chol/DOPE. Gene Ther 1995; 2: 603-613.
10. Labat-Moleur F et al. An electron microscopy study into the mechanism of gene transfer with lipopolyamines.
Gene Ther 1996; 3: 1010-1017.
11. Zabner J et al. Cellular and molecular barriers to gene transfer by a cationic lipid. J Biol Chem 1995; 270: 18997-19007.
12. Felgner JH et al. Enhanced Gene Delivery and Mechanism Studies With a Novel Series Of Cationic Lipid
Formulations. J Biol Chem 1994; 269: 2550-2561.
13. Sahenk Z et al. Gene Delivery to Spinal Motor Neurons. Brain Res 1993; 606: 126-129.
14. Iwamoto Y et al. Liposome-Mediated Bdnf Cdna Transfer In Intact and Injured Rat-Brain. Neuroreport 1996; 7: 609-612.
15. Roessler BJ, Davidson BL. Direct plasmid-mediated transfection of adult murine
brain-cells in-vivo using cationic liposomes. Neurosci Lett 1994; 167: 5-10.
16. Zhou X, Huang L. DNA transfection mediated by cationic liposomes containing lipopolylysine:
characterization and mechanism of action. Biochim Biophys Acta 1994; 1189:195-203.
17. Sorgi FL, Bhattacharya S, Huang L. Protamine sulfate enhances lipid-mediated gene
transfer. Gene Ther 1997; 4: 961-968.
18. Li S, Huang L. Protamine sulfate provides enhanced and reproducible intravenous
gene transfer by cationic liposome/DNA complex. Journal of Liposome Research 1997; 7: 207-219.
19. Vitiello L et al. Condensation of plasmid DNA with polylysine improves liposome-mediated gene transfer
into established and primary muscle cells. Gene Ther 1996; 3: 396-404.
20. Namiki Y, Takahashi T, Ohno T. Gene transduction for disseminated intraperitoneal
tumor using cationic liposomes containing non-histone chromatin proteins: cationic
liposomal gene therapy of carcinomatosa. Gene Ther 1998; 5: 240-246.
21. Gao X, Huang L. Potentiation of cationic liposome-mediated gene delivery by polycations.
Biochem 1996; 35: 9286-9286.
22. Fritz JD et al. Gene transfer into mammalian cells using histone-condensed plasmid DNA. Hum Gene Ther 1996; 7: 1395-1404.
23. Hagstrom JE et al. Complexes of non-cationic liposomes and histone H1 mediate efficient transfection
of DNA without encapsulation Biochim Biophys Acta 1996; 1284: 47-55.
24. Isaka Y et al. The HVJ liposome method. Exp-Nephrol 1998; 6: 144-147.
25. Gillock ET et al. Polyomavirus major capsid protein VP1 is capable of packaging cellular DNA when expressed
in the baculovirus system. J-Virol 1997; 71: 2857-2865 issn: 0022-2538x.
26. Chang D, Cai X, Consigli RA. Characterization of the DNA binding properties of
polyomavirus capsid proteins. Journal of Virology 1993; 67: 6327-6331.
27. Anderson CW, Young ME, Flint SJ. Characterization of the adenovirus 2 virion protein,
mu. Virology 1989; 172: 506-512.
28. Wood JN et al. Novel Cell-Lines Display Properties of Nociceptive Sensory Neurons. Proceedings of the Royal Society of London Series B-Biological Sciences 1990; 241: 187-194.
29. Budhrammahadeo V, Lillycrop KA, Latchman DS. The Levels of the Antagonistic Pou
Family Transcription Factors Bm- 3a and Bm-3b in Neuronal Cells Are Regulated in Opposite
Directions By Serum Growth-Factors. Neurosci Lett 1995; 185: 48-51.
30. Cooper RG et al. Polyamine analogues of 3 beta-[N-(N',N'-dimethylaminoethane)carbomoyl]cholesterol
(DC-Chol) as agents for gene delivery. Chemistry-α European Journal 1998; 4: 137-151.
31. McQuillin A et al. Optimization of liposome mediated transfection of a neuronal cell line. Neuroreport 1997; 8: 1481-1484.
32. Hosokawa K, Sung MT. Isolation and characterization of an extremely basic protein
from adenovirus type 5. Journal Of Virology 1976; 17: 924-934.
33. Caplen NJ et al. Liposome-Mediated Cftr Gene-Transfer to the Nasal Epithelium of Patients With Cystic-Fibrosis.
Nature Med 1995; 1: 39-46.
34. Nabel G, Chang A, Nabel E. Clinical Protocol: Immunotherapy of malignancy by in
vivo gene transfer into tumors. Hum Gene Ther 1992; 3: 399-410.
35. Nabel GJ et al. Direct gene-transfer with DNA liposome complexes in melanoma-expession, biological
activity, and lack of toxicity in humans. Proc Natl Acad Sci USA 1993; 90: 11307-11311.
36. Stewart MJ et al. Gene-transfer in vivo with DNA liposome complexes - safety and acute toxicity in
mice. Hum Gene Ther 1992; 3: 267-275.
37. Murray KD et al. DC-Chol/DOPE mediated transfections in differentiated sensory neurons. In preparation 1999.
38. Murray KD et al. Cationic liposome-mediated transfection in organotypic explant cultures. Gene Ther 1999; 6: 190-197.
39. Thierry AR et al. Characterization of liposome-mediated gene delivery: Expression, stability and pharmacokinetics
of plasmid DNA Gene Ther 1997; 4: 226-237.
40. Coonrod A, Li FQ, Horwitz M. On the mechanism of DNA transfection: efficient gene
transfer without viruses. Gene Ther 1997; 4:1313-1321.
41. Sebestyen MG et al. DNA vector chemistry: the covalent attachment of signal peptides to plasmid DNA Nat Biotechnol 1998; 16: 80-85.
42. Hagstrom JE et al. Nuclear import of DNA in digitonin-permeabilized cells. J Cell Sci 1997; 110 (Pt 18): 2323-2331.